Wounds are internal or external bodily injuries or lesions caused by physical means, such as mechanical, chemical, viral, bacterial, or thermal means, which disrupt the normal continuity of structures. Such bodily injuries include contusions, wounds in which the skin is unbroken, incisions, wounds in which the skin is broken by a cutting instrument, lacerations, and wounds in which the skin is broken by a dull or blunt instrument. Wounds may be caused by accidents or by surgical procedures.
The healing of wounds is a complex process involving a number of stages. These include; 1) coagulation, which begins immediately after injury; 2) inflammation, which begins a few minutes later; 3) a migratory and proliferative process (granulation stage), which begins within hours to days; and 4) a remodelling process with subsequent development of full strength skin (1-3).
Coagulation and Inflammation
Coagulation controls haemostasis and initiates healing by releasing a variety of growth factors and cytokines from degranulated platelets. During the inflammation phase, platelet aggregation and clotting form a matrix which traps plasma proteins and blood cells to induce the influx of various types of cells. Neutrophils are the first cells to arrive and function to phagocytise contaminating bacteria, digest the fibrin clot and release mediators to attract macrophages and activate fibroblasts and keratinocytes (3). Macrophages digest pathogens, debride the wound and secrete cytokines/growth factors (eg interleukin-1 (IL-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF)) that stimulate fibroblasts and endothelial cells. Overall, the inflammatory stage is important to guard against infection and promote the migratory and proliferative stages of wound healing.
Granulation and Remodelling of the Extracellular Matrix
These stages include cellular migration and proliferation. Although lymphocytes and macrophages are involved, the predominant cell types are epithelial, fibroblast and endothelial. Within hours of an injury, an epidermal covering, comprised mainly of keratinocytes, begins to migrate and cover the epidermis, a process known as re-epithelialisation. When they completely cover the wound they differentiate and stratify to form a new epidermis with a basal lamina. Angiogenesis (ie the formation of new blood vessels) occurs during this stage and provides nutrients for the developing tissue to survive. Fibroblasts migrate into the wound site and produce collagen and proteoglycans which ultimately give the wound tensile strength. As the remodelling phase progresses, granulation tissue is replaced by a network of collagen and elastin fibers leading to the formation of scar tissue.
Failed Wound Healing
Impaired dermal wound healing and/or dermal ulcers occur in patients with peripheral arterial occlusive disease, deep vein thrombosis, diabetes, pressure sores and burns (4). Despite intense investigation, the molecular mechanisms associated with impaired wound healing are poorly understood.
Wound healing is affected by numerous factors, including local factors (eg growth factors, edema, ischemia, infection, arterial insufficiency, venous insufficiency or neuropathy), systemic factors (eg inadequate perfusion and metabolic disease) and other miscellaneous factors, such as nutritional state, exposure to radiation therapy and smoking.
Leucocytes, particularly neutrophils, and macrophages persist in the surrounding tissue and secrete a range of proteases, including matrix metalloproteinases (MMPs) and serine proteases (5). Excessive accumulation of these enzymes interferes with the matrix remodelling (6). It is thought that agents which inhibit proteases will benefit wound healing (7). Another feature of some chronic wounds is the reduction or absence of angiogenesis, which prevents nutrients from accessing the newly formed tissue (8).
Existing Technologies to Improve Wound Healing
Chronic wounds are initially managed by treatment comprising eschar debridement, antibiotic treatment where appropriate, and regular dressing (2). Other dressings, such as hydrogels, hydrocolloids, or alginates, may also be used. Venous ulceration is treated by compression therapy, whereas arterial or diabetic ulcers require regular changes of dressings. Pressure sores are encouraged to heal by the relief of pressure at the injury site. Some other physical devices such as laser treatment, hyperbaric oxygen and electrical stimulation for arterial ulcers, are also used to promote wound healing (2, 9, 10).
For wounds that are unresponsive to such interventions, the use of tissue-engineered skin, such as Dermagraft or Apligraf, is an option. This therapy acts to prevent bacterial infection and allows the wound the chance to heal by normal reparative processes (11, 12). The use of such skin replacements to accelerate wound healing depends on the availability of an existing vascular supply in the existing wound.
Another approach to wound healing involves the administration of growth factors/cytokines, which have been shown to accelerate cell proliferation in vitro and/or to promote wound healing in some animal models. These include IL-1, platelet-derived growth factor (PDGF), EGF, VEGF, TGF-β, and bFGF (2). Procuren (Curative Technologies), an autologous platelet releasate, contains at least five growth factors, that aid in the formation of granulation tissue and re-epithelialisation. This autologous growth factor mix has achieved some success in human subjects with ulcerated limb lesions (13). However, on the whole, results from most clinical trials using growth factors/cytokines have been disappointing. For example, EGF failed to heal venous stasis ulcers and IL-1 failed to treat pressure sores effectively (2). Similar results were reported using bFGF (14). The reason for the lack of efficacy is not certain, but may relate to the multifactorial effects, some undesirable for healing, of growth factors/cytokines.
Thus, there is an ongoing need to identify and develop new agents for the promotion of wound healing.
Activated protein C (APC) is a serine protease having a molecular weight of about 56 kD that plays a central role in physiological anticoagulation. The inactive precursor, protein C, is a vitamin K-dependent glycoprotein synthesised by the liver and endothelium and is found in plasma. Activation of protein C occurs on the endothelial cell surface and is triggered by a complex formed between thrombin and thrombomodulin (15, 16). Another endothelial specific membrane protein, endothelial protein C receptor (EPCR), has been shown to accelerate this reaction more than 1000-fold (17). Endothelial APC functions as an anticoagulant by binding to the co-factor, protein S, on the endothelial surface, which inactivates the clotting factors Factor VIIIa and Factor Va. The importance of APC as an anticoagulant is reflected by the findings that deficiencies in this molecule result in familial disorders of thrombosis (18).
Recently, it has also been reported that APC additionally acts as an anti-inflammatory agent and directly activates the protease, gelatinase A (17, 20). Gelatinase A is secreted by many different cell types, including smooth muscle cells, fibroblasts and endothelial cells. By degrading the collagens present in the basement membrane (21) and allowing cells to invade the stroma, gelatinase A plays an important role in physiological remodelling and angiogenesis (22). Gelatinase A also plays an important role in numerous diseases, such as promoting the invasion of thymic epithelial tumors (23), promoting the destruction of the joint in arthritis by cleaving collagen from the cartilage matrix (24) and contributing to cardiac mechanical dysfunction during reperfusion after ischemia (25). In addition to its ability to degrade the matrix, gelatinase A can also target other substrates. For example, it cleaves big endothelin-1 to yield a potent vasoconstrictor, implicating gelatinase A as a regulator of vascular reactivity (26). Gelatinase A release can also mediate platelet aggregation (27).
Further, and as is demonstrated in the examples provided hereinafter, APC is also able to promote regeneration of endothelial cells after wounding in vitro, stimulate re-epithelialisation, fibroblast invasion and angiogenesis in a chicken embryo and enhance wound healing in a rat wounding model. These functions when taken together with the abovementioned anticoagulating, anti-inflammatory and Gelatinase A-activating functions, strongly indicate that APC, functional fragments thereof, and the precursor of APC (ie protein C) is/are useful for the treatment of wounds and, particularly, slow-healing wounds.